PROJECT ABSTRACT ! Vector-borne diseases affect millions of people worldwide and are becoming an increasing threat as the effects of climate change become more significant. Due to the rise in global temperatures, the likelihood of vector borne diseases will increase in many areas as the range of insects, such as mosquitos, expands and changes. Wolbachia are maternally transmitted bacteria that infect nearly half of the insect species on the planet and block the replication and transmission of several human pathogens within insect vectors (so called ?pathogen blocking?). Wolbachia-infected mosquitos have been released in several parts of the world in order to control the transmission of Dengue virus. Importantly, the mechanism of pathogen blocking is not well understood. Unfortunately, our understanding of the Wolbachia-vector systems have been limited due to the lack of genetic tools in Wolbachia and the intractability of many important vectors. Our project is aimed at overcoming these obstacles by using the model system, Drosophila melanogaster, to identify mechanisms used by Wolbachia to manipulate host cell biology. The strength and innovation of this proposal derives from the topic of interest, the combined expertise of two PIs, and the application of novel approaches. We will build upon a fruitful collaboration to identify and characterize the type IV secretion substrates in Wolbachia. In our first aim, we define the Wolbachia type IV translocation signal(s) and effector interactions with the Wolbachia VirD4 coupling protein. In our second aim, we take a complementary in vivo approach, identifying direct interactions between Wolbachia VirD4 and effectors during infection. Additionally, we test the hypothesis that Wolbachia secrete DNA via this T4SS and begin studies in eukaryotic model systems aimed at characterizing the function of these proteins. Our results will define the effectors used by Wolbachia during infection, revealing mechanisms used by the bacterium to manipulate host cell biology, allowing for precise experimentation in non- model organisms, such as mosquito vectors.